JP2011019446A - Lesion model animal of myodegeneration disease, and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a lesion model animal of myodegeneration disease, especially muscular dystrophy; to provide a method for producing the model animal; and to provide a method for screening a therapeutic agent or a prophylactic agent of the myodegeneration disease by using the lesion model animal. <P>SOLUTION: There is provided the method for producing the lesion model animal of the myodegeneration disease in which a dystrophin gene is knocked down or knocked out, and a hematogenous organ-type prostaglandin D synthetase (H-PGDS) is highly expressed. The method for screening the therapeutic agent or the prophylactic agent of the myodegeneration disease by using the lesion model animal of the myodegeneration disease is also provided. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a pathological model animal for muscle degenerative disease, particularly muscular dystrophy, and a method for producing the same, and a screening method for a therapeutic and / or preventive agent for muscle degenerative disease using the pathological model animal.

  A group of diseases accompanied by myopathy or myonecrosis is called myopathy, and typical diseases include muscular dystrophy and muscular atrophy. Duchenne muscular dystrophy, which has the largest number of patients among muscular dystrophies, is a disease in which one codon changes to a stop codon that means the end of protein synthesis due to a point mutation and dystrophin protein is not synthesized. It is a disease that affects only males due to sex chromosome recessive inheritance, and is said to be 3-5 per 100,000 population and 1 per 2000-3000 birth boys. Usually, patients with muscular dystrophy develop movement disorders around 3 to 5 years old, that is, abnormalities related to walking and standing such as being unable to run or falling, and become unable to walk around 10 years old. Later, spinal deformities or joint contractures progress rapidly and many patients develop respiratory failure, sometimes heart failure or pneumonia. There is still no good therapeutic means for muscular dystrophy, and the development of a therapeutic method is desired.

  In developing therapeutic drugs, a muscular dystrophy model that reflects human pathology is indispensable, and the therapeutic effects of drugs are investigated using mdx mice lacking the dystrophin gene and drug-induced muscle necrosis model animals (rats, mice). Yes.

  However, although mdx mice show a muscle necrosis pathology due to genetic abnormalities similar to those of Duchenne muscular dystrophy, they do not exhibit serious symptoms such as muscle atrophy, respiratory failure and heart failure unlike human pathologies. Furthermore, it has been reported that even when the dystrophin gene is knocked down in the local skeletal muscles of healthy mature mice to inhibit the synthesis of dystrophin protein, no significant myonecrosis is induced (Non-patent Document 1). In addition, muscle necrosis in drug-induced muscle necrosis model animals is transient, and long-term effects cannot be evaluated. Therefore, development of a rodent muscular dystrophy model animal that reflects human pathology is desired.

Hematopoietic prostaglandin D synthase (hereinafter referred to as H-PGDS) is expressed in microglia at the center, mast cells, Th2 lymphocytes, and Langerhans cells in the skin at the periphery. In addition, H-PGDS has been reported to be expressed in the necrotic muscle of Duchenne muscular dystrophy patients, to produce PGD ₂ locally, and to be strongly involved in the regulation of muscular dystrophy pathology (Non-patent Document 2). Furthermore, an inhibitor specific for H-PGDS has also been reported to significantly suppress the progression of muscular dystrophy pathology (Non-patent Document 3).

Hum Mol Genet. 17 (17), 2622-32 (2008) Acta Neuropathol. 104 (4), 377-84 (2002) Am J Pathol. 174 (5), 1735-44 (2009)

  In view of the above circumstances, the present inventors have conducted intensive studies in order to develop a rodent muscular dystrophy model animal that reflects human pathology. Further, the present inventors have found that the combination of inhibition of dystrophin protein synthesis by dystrophin gene deficiency (knockdown or knockout) in skeletal muscle and high expression of H-PGDS protein by introduction of H-PGDS gene markedly enhances myonecrosis pathology. It came to complete.

That is, the present invention relates to the following:
(1) A method for producing a disease state model animal for a muscle degenerative disease in which a dystrophin gene is knocked down or knocked out and a hematopoietic prostaglandin D synthase (H-PGDS) is highly expressed. :
(A) a step of knocking down or knocking out a dystrophin gene; and / or (b) a production method comprising a step of highly expressing H-PGDS;
(2) A disease state model animal of a muscle degenerative disease obtained by the method according to (1);
(3) The following steps:
(A) a step of administering a test drug to the animal according to (2);
(B) comparing the muscle degeneration in the animal according to claim 2 administered with the test drug in (a) and the muscle degeneration in the animal according to (2) not administered with the drug; and (c) the test When the muscle degeneration in the animal according to (2) to which the drug has been administered is suppressed more than the muscle degeneration in the animal according to claim 2 to which the drug has not been administered, the test drug is used as a therapeutic agent for a muscle degenerative disease and Or a screening method for a therapeutic agent and / or a prophylactic agent for muscle degenerative diseases, comprising a step of identifying as a prophylactic agent;
(4) The method according to (1), the animal according to (2), or the method according to (3), wherein the muscle degenerative disease is muscular dystrophy.

  According to the present invention, the effect of a therapeutic or prophylactic agent for muscular dystrophy can be determined in more detail as compared with conventional disease state animal models. Furthermore, according to the present invention, it is possible to discriminate molecules involved in pathogenesis and pathological progression in muscle degenerative diseases.

FIG. 1 shows GFP expression in gastrocnemius muscle 9 weeks after intramuscular injection of AAV-shDmd4 and AAV-hHPGDS constructs. Figure 2 shows the expression of GFP in gastrocnemius muscle 9 weeks after intramuscular injection of AAV-shDmd4 virus / saline construct FIG. 3 shows the expression of GFP in the gastrocnemius muscle 9 weeks after the intramuscular injection of the AAV-shCTRL virus / AAV-hHPGDS construct. FIG. 4 shows GFP expression in gastrocnemius muscle 9 weeks after intramuscular injection of AAV-shCTRL construct. FIG. 5 shows a comparison of GFP gene expression levels (after 8 weeks). FIG. 6 shows a comparison of human H-PGDS gene expression levels (after 8 weeks). FIG. 7 shows a comparison of dystrophin gene expression levels (after 4 weeks).

  That is, the present invention relates to a method for producing a disease state model animal for a muscle degenerative disease in which the dystrophin gene is knocked down or knocked out and H-PGDS is highly expressed in the first aspect. The animal of the present invention may be a rodent and may be produced using, for example, a mouse, rat, guinea pig, ferret, hamster, rabbit or the like. Examples of the muscular degenerative disease include muscular dystrophy, which includes Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, or Includes ocular muscular dystrophy.

  In the present invention, dystrophin gene expression may be altered by either knockdown or knockout. In addition, when knocking down the dystrophin gene, it is sufficient that the pathological condition of the degenerative disease of the muscular disease is shown in the animal obtainable by the method, and the expression of the dystrophin gene is compared with the expression level of the wild type homologous animal. It may be lowered as appropriate. Moreover, knockdown of the dystrophin gene should just be achieved in the whole body of the animal of this invention, or local parts, such as a skeletal muscle. In addition, in order to knock down or knock out the dystrophin gene, a known method may be used. For example, an adeno-associated virus (AAV) capable of efficient gene delivery may be used.

  In the present invention, high expression of H-PGDS may be achieved by a known method. For example, an H-PGDS gene operably linked to a promoter for high expression that can be used in an animal to be introduced is publicly known. This is achieved by introducing it into a cell in the manner described above. The high expression of H-PGDS only needs to indicate the pathological condition of a muscular disease degenerative disease in an animal obtainable by the method, and the expression of H-PGDS is appropriately compared with the expression level of a wild-type homologous animal. It only has to rise. High expression of H-PGDS may be achieved in the whole body of the animal of the present invention or locally such as skeletal muscle. The H-PGDS gene can be introduced by a known method, for example, an introduction system using AAV capable of efficient gene delivery.

  Only one or both of the knockdown or knockout of the dystrophin gene and the high expression of the H-PGDS gene in the animal of the present invention may be achieved. Furthermore, in the animal of the present invention, the expression of a specific gene other than dystrophin and H-PGDS may be increased or decreased systemically or locally in the animal of the present invention.

  In addition, as a pathological condition of the said muscular disease degenerative disease, the fall of the regeneration rate of the cell in a skeletal muscle is mentioned, for example, The fall of the regeneration rate of a cell may be attributed to acceleration | stimulation of muscle necrosis. In the present invention, the presence / absence and severity of a muscular disease degenerative disease may be determined from the appearance of the animal, but determined by methods known to those skilled in the art, such as molecular biological, histological or pathological means. May be.

  Moreover, this invention relates to the pathological condition model animal of the muscle degenerative disease manufactured in the 1st aspect in the 2nd aspect.

  Furthermore, the present invention, in the third aspect, relates to a screening method for a therapeutic agent and / or a preventive agent for myodegenerative diseases, using the animal model for pathological conditions of myodegenerative diseases. The screening method of the present invention includes, for example, (a) a step of administering a test drug to the animal produced in the first aspect; (b) muscle degeneration in the animal administered with the test drug in (a); Comparing the muscle degeneration in an animal produced in the embodiment and not administered the drug; and (c) the muscle degeneration in the animal administered the test drug is less than the muscle degeneration in the animal not administered the drug. If so, the method may comprise identifying the test agent as a therapeutic and / or prophylactic agent for myodegenerative diseases.

  The drug may be appropriately administered according to the animal condition, drug type, and administration method. The administration route of the drug may be any of intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, nasal administration, oral administration, and the like. In the present invention, the confirmation of the effect of the test agent may be determined from the appearance of the animal, but may also be determined by methods known to those skilled in the art, such as molecular biological, histological or pathological means.

  In a further aspect, the present invention relates to a substance for treating or preventing a muscle degenerative disease obtainable by the above screening method. In addition, such substances may be used in combination with any carrier or excipient for the purpose of treating or preventing muscle degenerative diseases.

  EXAMPLES The present invention will be described more specifically and in detail below with reference to examples. However, the examples are merely illustrative and do not limit the present invention.

Construction and purification of AAV8-shCTRL-hrGFP, AAV8-shDmd-hrGFP and AAV8-HPGDS constructs An H1 promoter is placed upstream of the CMV (cytomegalovirus) promoter of pUCAV-hrGFP (Stratagene), a pUC-based plasmid. Under the transcriptional control, shDmd, which is a short hairpin sequence for the purpose of knocking down dystrophin, or shCTRL as a negative control was arranged to construct pAAV-shDmd-hrGFP and pAAV-shCTRL-hrGFP. Moreover, pAAV-HPGDS was constructed by placing the human HPGDS gene under the control of the CMV promoter of pAAV-MCS (Stratagene), which is a pUC-based plasmid.

Recombinant AAV virions were produced in AAV293 cells (Stratagene) as follows. The AAV293 cells were cultured in complete DMEM (manufactured by Nacalai Tesque) (containing 4.5 g / L glucose, 10% non-immobilized fetal calf serum (FCS; GIBCO), and 2 mM glutamine). Subconfluent AAV293 cells were obtained from calcium phosphate precipitation (see, for example, J. Sambrook ET AL., “Molecular Cloning: A Laboratory Manual, Second Edition”, 1989, Cold Spring Harbor expression cassette, by A. Pold Harbor Laboratory). PAAV-shCTRL-hrGFP, pAAV-shDmd-hrGFP or pAAV-H-PGDS, pAAV-2 / 8 (including AAV rep and cap genes) and pHelper (including E2a, E4, and VA genes; Stratagene) 3 types of plasmids were simultaneously transfected. After 6 hours, the medium was replaced with complete DMEM medium, and further cultured at 37 ° C., 5% CO _{2 for} 72 hours. After the cultured cells were collected and centrifuged, the pelleted cells were freeze / thawed 4 times in Tris buffer (50 mM Tris / 150 mM NaCl, pH 8.5). In order to decompose contaminating genomic DNA, Benzonase (registered trademark) was added at a final concentration of 50 U / ml and reacted at 37 ° C. for 1 hour. The lysate was centrifuged at 7,000 g for 20 minutes, and the supernatant was collected.

  Each supernatant was subjected to density gradient centrifugation using Iodixanol (manufactured by Sigma Aldrich), and 40% Iodixanol fraction was collected. In order to exchange the solvent from Iodixanol to PBS, centrifugation was performed several times using an ultrafiltration membrane (Centricon 100K, manufactured by Millipore). The resulting virus solutions contain AAV8-shCTRL-hrGFP, AAV8-shDmd-hrGFP and AAV8-H-PGDS constructs, respectively (see Table 1).

これらのウイルス溶液をＰｒｏｔｅｉｎａｓｅＫ処理し、定量リアルタイムＰＣＲ法に供し、ベクター力価を決定した。

These virus solutions were treated with Proteinase K and subjected to quantitative real-time PCR to determine the vector titer.

Local gene knockdown and local high expression by in vivo administration of each vector The effect of dystrophin gene knockdown and H-PGDS overexpression on rat skeletal muscle was examined by efficient delivery of viral vectors.

  For Wistar male rats (4 weeks old, obtained from Japan SLC), each AAV construct prepared in Example 1 in the presence of isoflurane (1.5%) was injected into the hindlimb gastrocnemius muscle of rats. The combinations of constructs studied are shown below.

ジストロフィン遺伝子のノックダウンあるいはヒトＨ−ＰＧＤＳ過剰発現による表現型を調べるために、各コンストラクトをラットの腓腹筋に注射の２週間、４週間、６週間および８週間後にサンプリングを行った。サンプリング方法は、以下の通りである。

In order to examine the phenotype due to knockdown of dystrophin gene or overexpression of human H-PGDS, each construct was sampled at 2, 4, 6, and 8 weeks after injection into rat gastrocnemius muscle. The sampling method is as follows.

  Rats were euthanized by intraperitoneal administration of an excess amount of pentobarbital (dose 100 mg / kg), the whole body was perfused with PBS, and the gastrocnemius muscle was removed. The extracted tissue was immediately frozen in isopentane cooled to about -70 ° C. Frozen tissues were stored frozen at -80 ° C until examination of RNA extraction, Western blotting, and immunohistochemical staining. Alternatively, an excessive amount of pentobarbital is administered intraperitoneally (dose 100 mg / kg) to be euthanized, and the whole body is perfused with PBS, followed by perfusion fixation with a neutral buffered 10% formalin solution (manufactured by Sigma-Aldrich). The tissue was later removed and equilibrated with 20% sucrose and stored at 4 ° C.

  An unfixed frozen sample or a sample after formalin fixation was placed in a cryostat, and sections of 5 to 10 microns (μm) were collected. A part of the sample was subjected to hematoxylin / eosin staining, immunohistochemical staining, or fluorescent staining using green fluorescent protein (GFP) as an index.

Analysis by PCR Using a rat tissue injected with saline intramuscularly as a negative control, the gene expression fluctuation in the gastrocnemius muscle was examined by quantitative PCR. As a result, the dystrophin gene was significantly detected after 4 weeks of AAV administration (infection). A significant decrease in expression was detected. On the other hand, the expression of the human H-PGD synthase gene was detected from 2 weeks after AAV administration (infection) and persisted until 8 weeks.

Analysis by fluorescent staining using green fluorescent protein (GFP) as an index The GFP gene as an index of AAV infection was detected from 2 weeks after AAV administration (infection) and persisted until 8 weeks.

Analysis by immunohistochemical staining Thin sections of 5-10 μm were washed with PBS and treated with 0.3% H ₂ O ₂ for 30 minutes to block endogenous peroxidase activity. Washed again with PBS, followed by 1 hour incubation at room temperature in blocking solution (10% goat serum and 0.01% Triton-X100 in PBS). Samples were then incubated overnight at 4 ° C. with anti-dystrophin-mouse monoclonal antibody (Novokastra) (1: 100) or anti-human H-PGDS-mouse monoclonal antibody (1: 50000). Washed 3 times in PBS, incubated with biotinylated goat anti-mouse IgG (Vector) (1: 200) for 1 hour at room temperature and washed again with PBS. Antibody binding was visualized using streptavidin horseradish peroxidase (1: 300).

  When each construct was injected into skeletal muscle and analyzed by immunohistochemical staining after 4 weeks, dystrophin protein was not detected or decreased in gastrocnemius muscle cells (muscle fibers) in groups 1, 2 and 5. It was. On the other hand, in groups 3 and 4, dystrophin protein was detected in gastrocnemius muscle cells (muscle fibers) as in the untreated group (group 6) in which AAV treatment was not performed. In groups 1, 3 and 4, human H-PGDS protein was detected in gastrocnemius muscle cells (muscle fibers). However, in groups 3 and 4, as in the untreated group (group 6) in which AAV treatment was not performed, human H-PGDS protein was not detected in gastrocnemius muscle cells (muscle fibers).

  Based on the above results, knockdown of the dystrophin gene and overexpression of the human-H-PGDS gene are achieved after 4 weeks at the latest after injection (infection) of the AAV construct, and the effect is sustained for 8 weeks or more. It was confirmed.

  Furthermore, histological examination of gene expression 9 weeks after the injection (infection) of the AAV construct was performed using the fluorescence intensity of the GFP protein as an index. As a result, dystrophin gene knockdown and human-H-PGDS gene high expression were aimed. Only the group injected with the construct (Group 1) showed strong GFP protein expression. On the other hand, in the other groups, the expression of GFP protein was decreased.

  According to the present invention, it is possible to determine the effect of a therapeutic agent or a preventive agent on a muscle degenerative disease such as muscular dystrophy in more detail as compared with a conventional disease state model animal, and development of a more efficient therapeutic agent and preventive agent Is possible. Furthermore, according to the present invention, it is possible to discriminate molecules involved in pathogenesis and pathological progression in muscle degenerative diseases.

SEQ ID NO: 1 ； short hairpin sequence for dystrophin gene knockout

Claims (4)

A method for producing a pathological model animal for a muscle degenerative disease in which a dystrophin gene is knocked down or knocked out and a hematopoietic prostaglandin D synthase (H-PGDS) is highly expressed, comprising the following steps:
(A) a step of knocking down or knocking out a dystrophin gene; and / or (b) a production method comprising a step of highly expressing H-PGDS.   A pathological model animal of a muscle degenerative disease obtained by the method according to claim 1. The following process:
(A) a step of administering a test drug to the animal according to claim 2;
(B) comparing the muscle degeneration in the animal of claim 2 administered with the test drug in (a) with the muscle degeneration in the animal of claim 2 not administered the drug; and (c) test 3. A therapeutic agent for a muscle degenerative disease, wherein muscle degeneration in an animal according to claim 2 to which a drug is administered is suppressed as compared to muscle degeneration in an animal according to claim 2 to which the drug is not administered. Alternatively, a method for screening a therapeutic agent and / or prophylactic agent for muscle degenerative diseases, comprising a step of identifying as a prophylactic agent.   The method according to claim 1, the animal according to claim 2, or the method according to claim 3, wherein the muscle degenerative disease is muscular dystrophy.

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